This paper is an investigation to compare the in vivo controlled release of a plasmid DNA from biodegradable hydrogels prepared from gelatin cationized by different amine compounds, ethylenediamine, putrescine, spermidine, and spermine and the consequent profile of gene expression. Cationized gelatin prepared through the chemical introduction of each amine compound was crosslinked by various concentrations of glutaraldehyde to obtain cationized gelatin hydrogels for the carrier of plasmid DNA release. When the cationized gelatin hydrogels incorporating 125I-labeled plasmid DNA were implanted into the femoral muscle of mice, the radioactivity remaining decreased with time and the retention period of radioactivity prolonged with a decrease in the water content of hydrogels. When 125I-labeled cationized gelatin hydrogels with the higher water content was implanted, the radioactivity remaining was decreased faster with time. The remaining time profile of plasmid DNA radioactivity was in good accordance with that of hydrogel radioactivity, irrespective of the type of cationized gelatin. Following intramuscular implantation, any cationized gelatin hydrogel incorporating plasmid DNA enhanced the expression level of plasmid DNA to a significantly higher extent than the free plasmid DNA injection. In addition, prolonged time period of gene expression was observed although there was no significant difference in the expressed period between the cationized gelatin hydrogels. It was concluded that plasmid DNA of biological activity was released from every cationized gelatin hydrogel accompanied with the in vivo degradation, resulting in enhanced and prolonged gene expression.